Histoplasma capsulatum var. farciminosum, the causative agent of epizootic lymphangitis (EZL), is endemic in parts of Africa. Diagnosis based on clinical signs and microscopy lacks specificity and is a barrier to further understanding this neglected disease. Here, a nested PCR method targeting the internal transcribed spacer (ITS) region of the rRNA operon was validated for application to equine clinical samples. Twenty-nine horses with signs of EZL from different climatic regions of Ethiopia were clinically examined. Blood samples and aspirates of pus from cutaneous nodules were taken, along with blood from a further 20 horses with no cutaneous EZL lesions. Among the 29 horses with suspected cases of EZL, H. capsulatum var. farciminosum was confirmed by extraction of DNA from pus and blood samples from 25 and 17 horses, respectively. Positive PCR results were also obtained with heat-inactivated pus (24 horses) and blood (23 horses) spotted onto Whatman FTA cards. Two positive results were obtained among blood samples from 20 horses that did not exhibit clinical signs of EZL. These are the first reports of the direct detection of H. capsulatum var. farciminosum in equine blood and at high frequency among horses exhibiting cutaneous lesions. The nested PCR outperformed conventional microscopic diagnosis, as characteristic yeast cells could be observed only in 14 pus samples. The presence of H. capsulatum var. farciminosum DNA was confirmed by sequencing the cloned PCR products, and while alignment of the ITS amplicons showed very little sequence variation, there was preliminary single nucleotide polymorphism-based evidence for the existence of two subgroups of H. capsulatum var. farciminosum. This molecular diagnostic method now permits investigation of the epidemiology of EZL.

Temporal temperature gradient electrophoresis (TTGE) analysis of 16S rRNA gene fragments amplified with primers selective for eubacteria and beta-proteobacterial ammonia-oxidising bacteria (AOB) was used to analyse changes in bacterial and AOB community profiles of an upland pasture following soil improvement treatments (addition of sewage sludge and/or lime). Community structure was compared with changes in activity assessed by laboratory measurements of basal respiration and ammonia oxidation potentials, and with measurements of treatment- and time-related changes in soil characteristics. The predominant bacterial populations had a high degree of similarity under all treatment regimens, which was most pronounced early in the growing season. Most of the differences that occurred between soil samples with time could be accounted for by spatial and temporal variation; however, analysis of variance and cluster analysis of similarities between 16S rDNA TTGE profiles indicated that soil improvement treatments exerted some effect on community structure. Lime application had the greatest influence. The impact of soil improvement treatments on autotrophic ammonia oxidation was significant and sustained, especially in soils which had received sewage sludge and lime treatments in combination. However, despite obvious changes in soil characteristics, e.g. pH and soil nitrogen, increasing heterogeneity in the AOB community structure over time obscured the treatment effects observed at the beginning of the experiment. Nevertheless, time series analysis of AOB TTGE profiles indicated that the AOB community in improved soils was more dynamic than in control soils where populations were found to be relatively stable. These observations suggest that the AOB populations exhibited a degree of functional redundancy.

Reasons for performing study Histoplasma capsulatum var. farciminosum (HCF), causing epizootic lymphangitis (EZL), is endemic in parts of Africa including, Ethiopia, Senegal and Gambia. Despite its high prevalence, impact on animal welfare and socio‐economic importance, there is little evidence upon which to build practical disease control strategies. The performance and availability of diagnostic tests currently used by clinicians is problematic. Methods such as pattern recognition of clinical signs and microscopy lack specificity and other reported methods are either not commercially available or not readily feasible in these settings (e.g. culture). This is a significant barrier to further understanding this disease within endemic countries. Objectives To validate a nested PCR method to confirm the presence of HCF in equine clinical samples. Study design Cross‐sectional. Methods Twenty‐nine horses with suspected EZL were included from topographically varied regions of Ethiopia. Clinical data, lesion location drawn onto equine silhouettes, blood samples and aspirates of pus from cutaneous nodules were obtained before treatment provided by SPANA clinic. Blood and clinical data were collected from a further 20 horses with no cutaneous EZL lesions. Giemsa stained impression smears of pus were examined microscopically. Aliquots of heat‐inactivated pus and blood were inoculated onto Whatman FTA cards and imported to the UK with Defra approved licensing. A nested PCR targeting the ITS region, was used to identify samples containing HCF and PCR products were sequenced. Results HCF was confirmed in heat‐inactivated FTA card pus samples from 24 horses, additionally, 23 blood samples were positive from EZL suspected cases. Bioinfomatic analyses suggested that there was diversity within the ITS region among these HCF products. Conclusions These PCR techniques allow the rapid diagnosis of HCF directly from equine clinical samples. The identification of HCF in blood raises questions about the pathogenesis of HCF in horses and warrants further investigation. Acknowledgements We thank the SPANA Ethiopia team; participating cart‐horse owners; the Ethio‐Belgian project; Addis Ababa University; Gabrielle Laing and the PHE UK Mycology reference laboratory. Ethical animal research: Ethical approval for the project was awarded from the University of Liverpool and The College of Veterinary Medicine and Agriculture, Addis Ababa University. Sources of funding: SPANA UK (registered charity), the Institute of Infection and Global Health, University of Liverpool and an Sfam studentship. Competing interests: Dr Stringer was veterinary director at SPANA while this project was conducted and provided consultative and logistical input.

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